Fibronectins, their fibrillogenesis, and in vivo functions

Abstract

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN-FN and cell-FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.

Figure 1.

FN domain organization and isoforms. Each FN monomer has a modular structure consisting of 12 type I repeats (cylinders), 2 type II repeats (diamonds), and 15 constitutive type III repeats (hexagons). Two additional type III repeats (EIIIA and EIIIB, green) are included or omitted by alternative splicing. The third region of alternative splicing, the V region (green box), is included (V120), excluded (V0), or partially included (V95, V64, V89). Sets of modules comprise domains for binding to other extracellular molecules as indicated. Domains required for fibrillogenesis are in red: the assembly domain (repeats I_1-5) binds FN, III_9-10 contains the RGD and synergy sequences for integrin binding, and the carboxy-terminal cysteines form the disulfide-bonded FN dimer (‖). The III_1-2 domain (light red) has two FN binding sites that are important for fibrillogenesis. The amino-terminal 70-kDa fragment contains assembly and gelatin-binding domains and is routinely used in FN binding and matrix assembly studies.

Figure 2.

FN matrices in culture and in vivo. (A) Indirect immunofluorescence image shows a typical FN matrix assembled by cells in monolayer culture. Dexamethasone-treated HT1080 fibrosarcoma cells (Brenner et al. 2000) were fixed and permeabilized, and FN was visualized by staining with anti-FN antibodies and fluorescein-tagged secondary antibodies. Fibrils (green) extend around and between the cells whose nuclei are visualized by staining DNA with DAPI (blue). Intracellular staining adjacent to the nuclei is most likely FN that is trafficking through the secretory pathway. The organization of fibrils assembled by fibroblast-like cells (A) differs from the fibril arrangements formed by blastocoel roof cells (B) which grow in a multilayered sheet. (B) Indirect immunofluorescence image of the blastocoel roof from a mid-late gastrula stage Xenopus laevis embryo shows native assembly state of the FN matrix at this stage. This is an en face view of the innermost layer of cells in the multicell layered blastocoel roof. These cells face the fluid-filled blastocoel and assemble FN fibrils on their free surfaces. FN fibrils (green) extend across surfaces of cells outlined by C-cadherin staining (blue). Cortical assembly of actin (red) is required for the formation of FN fibrils from pericellular FN that initially assembles at cell–cell boundaries. Note the apparent incomplete progression, or delay, of FN fibril assembly by some cells in the layer. These are cells that have entered the layer as gastrulation proceeds and as a consequence of radial intercalation. Once resident in this layer these cells begin to assemble matrix on their newly “free” surfaces. (Scale bars, 25 µm.)